In all eukaryotes, actin is assembled into highly dynamic thin filaments, which are organized into networks that provide polarity and force to drive different cellular processes (e.g., cell migration, cytokinesis, muscle contraction, and endocytosis). The proper regulation of actin assembly and actin cytoskeletal function is impaired in many disease states, particularly those relevant to heart, lung, and blood research. Mutations in human WASp (Wiskott Aldrich Syndrome protein), a stimulator of actin related protein (Arp) 2/3 complex-medicated actin assembly, cause defects in neutrophil cell motility and loss of immune function. In asthma, calcium levels rise in response to histamine release and induce smooth muscle contraction in respiratory tracts of the lungs. Defects in actin itself are linked directly to dilated cardiomyopathy and heart failure, and mutations in key actin regulators (e.g., cofilin) lead to muscle denervation and dystrophy. Thus, understanding the biochemical basis of actin assembly is an important first step in defining how these pathological processes disrupt normal function in these tissues. Studies in the budding yeast Saccharomyces cerevisiae have been instrumental in dissecting the functions of key actin regulators, because combined genetic and biochemical approaches can be used. The objectives of this research career award (RCA) are to expand the specific aims of an existing R01 (Regulation of Actin Assembly in Budding Yeast) by introducing two new microscopy tools to study the structure and mechanism of action of Arp2/3 complex. Total internal reflection fluorescence (TIRF) microscopy will be used to study actin filament polymerization and branching by Arp2/3 complex and its regulation by WASp, Abp1, and coronin in real time. Electron microscopy and single particle image analysis will be used to study the structures of mutant Arp2/3 complexes with impaired activities and in vivo defects. The funding of this proposal would significantly enhance the ability of the PI to accomplish these goals by reducing his teaching and administrative duties. The interdisciplinary nature of this research program necessitates central involvement of the PI in training of students and postdocs in techniques and areas in which they are inexperienced. The PI will be directly involved in the integration of TIRF microscopy and single particle imaging into his research program.